1. Field of the Invention
The present invention relates generally to the field of virology. More specifically, the present invention provides assays that measure virus binding and entry into target cells.
2. Description of the Related Art
pH-dependent models such as influenza A and semliki forest virus have been used to study the mechanisms of enveloped virus entry because it is possible to induce en-masse fusion event by dropping the pH of the medium. Fluorescence dequenching or FRET assays are used to measure the kinetics of fusion in such viruses and have been used to understand the effects of mutations and anti-viral drugs. In this assay, fluorescent probes incorporated in the virus membranes mix with and become diluted in the target cell or liposome membrane. The resulting change in fluorescence gives a real-time measure of fusion. These entry assays cannot be applied easily to pH-independent viruses since fusion events are infrequent, cannot be coordinated and the receptors are difficult to manipulate since they tend to be integral, multi-transmembrane span-containing proteins. In such studies, passive diffusion of fluorophore contributes significantly to the signal and complex analysis of the data is required to observe signal due to fusion.
The need for more sensitive measurement of pH-dependent virus entry has led to the development of assays to detect cell-cell fusion, early genome replication events and assays that use recombinant viral protein-GFP fusions. In assays detecting cell-cell fusion, which is used to confirm the role of factors important in fusion, cells made to express virus envelope proteins on the surfaces are labeled with one fluorophore and mixed with target cells bearing receptor and second fluorophore. Fusion is measured by observing syncytia formation. Independent labeling of cell membrane and cytoplasm provides information on membrane and cytoplasm mixing. It has been shown that syncytia formation is slow and does not correlate to infection kinetics. Additionally, in case of HIV, the chemokine receptor, Bonzo promotes syncytia but does not play any significant role in entry.
There are several assays that detect virus infection. Some assays measure infection by using reporter gene expression in the infected cell. This is a very complicated process. To obtain expression, a virus must penetrate the cell membrane, the core must be trafficked to the correct subcellular location, then the genome is exposed and finally the reporter is expressed. This requires, for example in retroviruses, the cell to be at a specific stage and the gene expression requires at least 24 hours after contact with cells which is far removed from the initial entry event.
Other assays, which involve making virus-protein fusions to green fluorescent protein, have been useful to follow virus after it has entered the cell. The use of fluorescently labeled dUTP even permits visualization of genomes undergoing reverse transcription. These assays cannot be easily used to examine virus entry as cell bound viruses cannot be differentiated from those which have just entered the cells. For retroviruses, particle to infectious particle ratios typically exceed 10-100. This means that most viruses are either defective or trafficked to non-productive pathways within the cell.
Contents-mixing assays, which measure release of virus contents into the cell or target vesicle, demonstrate great potential for rapid measurement of virus entry. In case of retroviruses, it takes advantage of the fact that viral cDNA synthesis is limited by access to deoxyribonucleotides. When the viral genome is exposed by capsid disassembly after entry, dNTPs can access the viral polymerase and synthesis proceeds. Transcripts can then be detected by PCR, typically around 4 hours after cell contact. It is not known at what point within this 4-hour window does genome uncoating take place nor is it known when the dNTP pool is contacted.
A method where enzyme β-lactamase is fused to HIV protein vpr was developed recently to obtain quantitative data. Vpr is packaged into HIV particles as part of virus assembly and provides a means of targeting a marker enzyme into the particle. Caged substrate was perfused into cells to give signal. Detection of entry required 12 hours of cell culture for production of sufficient reaction product. To enable shorter measurement times, an MOI of more than 10-100, which is not physiological, is required.
A few virus entry inhibitors exist for influenza and HIV. While these inhibitors show some promise, they are far from perfect and have low efficacy. Discovery of similar but more effective drugs has been hindered by a lack of high-throughput, high signal to noise assay for screening lead compounds/drugs. Thus, the prior art is deficient in assays that are fast, simple, physiological and sensitive to measure virus entry. The present invention fulfils this need by providing methods that allow rapid and high-throughput non-radioactive detection of virus entry.